Wnt signaling regulates hepatocyte cell division by a transcriptional repressor cascade

Significance As a general model for cell cycle control, repressors keep cells quiescent until growth signals remove the inhibition. For S phase, this is exemplified by the Retinoblastoma (RB) protein and its inactivation. It was unknown whether similar mechanisms operate in the M phase. The Wnt signaling pathway is an important regulator of cell proliferation. Here, we find that Wnt induces expression of the transcription factor Tbx3, which in turn represses mitotic inhibitors E2f7 and E2f8 to permit mitotic progression. Such a cascade of transcriptional repressors may be a general mechanism for cell division control. These findings have implications for tissue homeostasis and disease, as the function for Wnt signaling in mitosis is relevant to its widespread role in stem cells and cancer.

Cell proliferation is tightly controlled by inhibitors that block cell cycle progression until growth signals relieve this inhibition, allowing cells to divide. In several tissues, including the liver, cell proliferation is inhibited at mitosis by the transcriptional repressors E2F7 and E2F8, leading to formation of polyploid cells. Whether growth factors promote mitosis and cell cycle progression by relieving the E2F7/E2F8-mediated inhibition is unknown. We report here on a mechanism of cell division control in the postnatal liver, in which Wnt/β-catenin signaling maintains active hepatocyte cell division through Tbx3, a Wnt target gene. The TBX3 protein directly represses transcription of E2f7 and E2f8, thereby promoting mitosis. This cascade of sequential transcriptional repressors, initiated by Wnt signals, provides a paradigm for exploring how commonly active developmental signals impact cell cycle completion. liver j Wnt j proliferation Cells in adult tissues remain mostly quiescent unless they become activated by growth factors to enter the cell cycle and proliferate (1). Quiescence is commonly imposed by inhibitors that prevent cells from entering S phase (2). To allow cell cycle entry, growth factors repress these inhibitors and activate the G1/S transition (3). In some tissues, including the liver, cells can enter S phase to duplicate their genome, but do not complete M phase, thus becoming polyploid (4)(5)(6). In the liver, two members of the E2F family of cell cycle regulators, E2f7 and E2f8, are responsible for this form of cell cycle arrest (7,8). Expression of E2f7 and E2f8 is induced at the time of weaning when the tissue switches from initially containing diploid hepatocytes to becoming mostly polyploid (4,9,10). Acting as mutually redundant transcriptional repressors, E2F7 and E2F8 inhibit the expression of many genes, including Aurka/b, Ccnb1, and Plk1, which encode proteins that act during mitotic progression (7,8,11). Deleting E2f7 and E2f8 from hepatocytes results in completion of M phase and the generation of diploid daughter cells (7,8). Therefore, in the liver, DNA synthesis is uncoupled from cell proliferation due to the activity of E2F7 and E2F8. Whether growth factors can suppress the activity of E2f7 and E2f8 and thereby promote cell division is currently not known.
Hepatocytes in the postnatal liver expand rapidly in a Wnt/β-catenin signalingdependent way (12)(13)(14)(15)(16). In the mouse and human liver, Wnt ligands are produced by endothelial cells of the central vein branches and the nearby sinusoids (17)(18)(19), creating a zonated expression pattern of Wnt target genes and key metabolic enzymes in pericentral hepatocytes (20)(21)(22). Among the genes highly expressed in the pericentral zone is the transcriptional repressor Tbx3 (17,19). Tbx3 is required for embryonic liver development and promotes hepatic progenitor proliferation likely by repressing the p19 ARF (Cdkn2a) cell cycle inhibitor (23). Tbx3 also maintains expression of hepatocyte-lineage genes, such as Cebpα and Hnf4α, and represses the cholangiocyte fate (23,24). In hepatic tumor cells with activating β-catenin mutations, Tbx3 is overexpressed and mediates cell proliferation and survival (25). These findings highlight the roles of Tbx3 in hepatic cell cycle control during liver development and tumorigenesis.
In the work presented here, we have studied the regulation of hepatocyte cell division in vivo during postnatal liver growth as well as in primary cell culture. We report that Wnt growth factors induce Tbx3 expression, which subsequently represses E2f7 and E2f8 to regulate cell division. We conclude that Wnt signals regulate the cell cycle at the level of mitosis through this cascade of repressive interactions.

Results
Tbx3 Promotes Hepatocyte Proliferation Downstream of Wnt Signaling. Similar to the adult liver, Tbx3 is expressed in Wnt-responsive pericentral hepatocytes during postnatal liver growth (SI Appendix, Fig. S1A). To determine whether Tbx3 is a target of Wnt signaling in the liver, we either overactivated or eliminated the Wnt pathway in the tissue,

Significance
As a general model for cell cycle control, repressors keep cells quiescent until growth signals remove the inhibition. For S phase, this is exemplified by the Retinoblastoma (RB) protein and its inactivation. It was unknown whether similar mechanisms operate in the M phase. The Wnt signaling pathway is an important regulator of cell proliferation. Here, we find that Wnt induces expression of the transcription factor Tbx3, which in turn represses mitotic inhibitors E2f7 and E2f8 to permit mitotic progression. Such a cascade of transcriptional repressors may be a general mechanism for cell division control. These findings have implications for tissue homeostasis and disease, as the function for Wnt signaling in mitosis is relevant to its widespread role in stem cells and cancer. and used Glutamine synthetase (GS), a Wnt target (26), to assess pathway activity. Using CRISPR-Cas9 gene editing, we deleted Apc, a negative regulator of Wnt signal transduction in hepatocytes, and observed ectopic expression of Tbx3 (Fig. 1A). Conversely, mice carrying mutations for the Wnt secretion machinery gene Wntless (Wls) exhibited a partial loss of Tbx3 expression (Fig. 1A). These findings agree with Tbx3 acting as a target of Wnts in other contexts (25,(27)(28)(29)(30).
To examine the function of Tbx3 in hepatocytes under defined conditions, we employed a recently developed method for long-term culture and genetic manipulation of primary hepatocytes, where cells multiply rapidly in a Wnt-dependent manner (31). We knocked down Tbx3 in cultured hepatocytes using two different shRNA constructs (SI Appendix, Fig. S1B). This resulted in significant slowing down of hepatocyte proliferation (Fig. 1B) and increased numbers of polyploid cells ( Fig. 1C and SI Appendix, Fig. S1C and Table S1), suggesting that hepatocytes failed to complete M phase. Conversely, overexpression of Tbx3 in hepatocytes was sufficient to increase proliferation ( Fig.  1D and SI Appendix, Fig. S1D and E), even in the absence of CHIR99021, a GSK3 inhibitor that activates Wnt signaling (Fig. 1D). Additionally, overexpression of Tbx3 increased the percentage of diploid cells ( Fig. 1E and SI Appendix, Table S1). As expected, CHIR99021 alone enhanced proliferation of hepatocytes albeit not to the same degree as Tbx3 overexpression (Fig. 1D).
Deletion of Tbx3 and Changes in Ploidy. We examined Tbx3 function in the growing postnatal liver by generating inducible Tbx3 loss-of-function mutant mice (Tbx3 knockout [KO]) carrying the Axin2-rtTA; TetO-H2B-GFP; TetO-Cre; Tbx3 f/f genotype. Axin2-rtTA is a doxycycline-inducible transgene, which leaves the C B endogenous Axin2 gene intact and is expressed in a pattern similar to Tbx3 ( Fig. 2A) (17). To obtain maximal elimination of Tbx3, doxycycline was administered from postnatal week 2 to week 4, at which point tissues were harvested for analysis (Fig. 2B). Nuclear size measurement ( Fig. 2C) and nuclear DNA content analysis ( Fig. 2D) of GFP-labeled hepatocytes revealed a significant increase in the proportion of polyploid nuclei in Tbx3 KO livers (>2N: 59.8% in control and 92% in Tbx3 KO) (SI Appendix, Table S1). The decrease in diploidy and the concomitant increase in polyploidy indicated that in the absence of Tbx3, hepatocytes were able to complete S phase but not M phase. In parallel, we used the inducible and hepatocyte-specific Albumin-CreERT2 (Alb-CreERT2) driver to eliminate Tbx3 in all hepatocytes. We administered a single dose of tamoxifen at postnatal day 3 (P3) and analyzed the livers at different timepoints into adulthood ( Fig. 2E and SI Appendix, Fig. S2A and B). Loss of Tbx3 did not affect liver shape or relative mass, at any timepoint (SI Appendix, Fig. S2C and D). Consistent with the observations above, Tbx3 KO livers were comprised of larger nuclei at all analyzed timepoints compared to control ( Fig. 2F and G) and DNA content analysis confirmed increased ploidy in mutant tissues ( Fig. 2H and SI Appendix, Table S1). Taken together, we conclude that   Tbx3 regulates polyploidization of hepatocytes in vivo and in vitro by its control over cell division.
TBX3 Represses Transcription of E2f7 and E2f8. To identify mechanisms of Tbx3 function, a known repressor of transcription, we investigated potential target genes of TBX3 using chromatin immunoprecipitation-sequencing (ChIP-Seq) in Tbx3-overexpressing mouse primary hepatocytes. We found that the TBX3 protein binds to promoter and enhancer regions of E2f7 and E2f8, respectively, and verified this interaction by ChIP-qPCR ( Fig. 3A and B). This suggested that TBX3 represses E2f7 and E2f8, and indeed E2f7 and E2f8 transcripts were both ectopically increased in Tbx3 KO livers ( Fig. 3C and D). Knockdown and overexpression of Tbx3 in cultured primary hepatocytes further corroborated the interactions with E2f7 and E2f8 ( Fig. 3E and F).
To verify that repression of E2f7 and E2f8 by TBX3 occurs on the regulatory sequences of these genes, we employed a luciferase reporter assay in human hepatoblastoma HepG2 cells, which express TBX3 at high levels (32). Addition of the mouse E2f7 or E2f8 enhancer regions containing the TBX3 binding sites to an HSV-TK reporter construct led to significant repression of the luciferase reporter activity compared to control vector with HSV-TK promoter only (Fig. 3G). Moreover, reporter activity was increased when TBX3 was knocked down or T-box binding motifs were mutated or deleted from the enhancer regions, confirming that TBX3 is a specific transcriptional repressor of E2f7 and E2f8 (Fig. 3G and SI Appendix, Fig. S3A). In the adult human liver, TBX3 is specifically expressed in pericentral hepatocytes, similar to the mouse liver (SI Appendix, Fig. S3B). Analysis of ChIP-Seq data from the ENCODE database (GSE105374)    (33,34) showed that human TBX3 protein also binds to E2F7 and E2F8 enhancers (SI Appendix, Fig. S3C), suggesting that TBX3 has conserved functions in both human and mouse livers. Hence, a variety of different experiments provided evidence that TBX3 directly regulates E2F7 and E2F8 expression.
Epistatic Relationships between Tbx3 and E2f7/E2f8. The negative interactions between Tbx3 and E2f7/E2f8 and the up-regulation of E2f7 and E2f8 in the absence of Tbx3 would imply that loss of all three genes would suppress the Tbx3 knockout phenotype. To test possible epistatic interactions by mouse genetics, we generated triple conditional mutants of Tbx3, E2f7, and E2f8 (Tbx3-E2f7-E2f8 TKO). First, we used the Axin2-rtTA; TetO-GFP; TetO-Cre system to delete all three genes by continuously administering doxycycline from postnatal week 2 to week 4 (Fig. 4A). GFP-labeled nuclei in Tbx3-E2f7-E2f8 TKO livers were indistinguishable from controls in ploidy and size (Fig. 4B-D and SI Appendix, Table S1). Similar results were obtained in the pan-hepatocyte deletion model of Tbx3-E2f7-E2f8 TKO livers (SI Appendix, Fig. S4A-C and Table S1). These findings show that simultaneous removal of E2f7, E2f8, and Tbx3 resolves the polyploid phenotype caused by removal of Tbx3 alone. These genetic interactions imply a linear pathway whereby Wnt activates Tbx3, which represses the mitotic inhibitors E2f7 and E2f8; this therefore relieves cell cycle arrest and maintains hepatocytes in a diploid, dividing state.
Loss of Tbx3 Leads to Fibrosis. Despite the functions attributed here to Tbx3 in postnatal liver growth, we did not observe major changes in liver weight or morphology (SI Appendix, Fig. S2C and D). To identify physiological impact of long-term loss of Tbx3 on the liver, we ablated the gene in neonates with the Alb-CreERT2 driver and aged the animals to adulthood (Fig. 5A). Upon histological analysis, we observed cytoplasmic vacuoles and anomalies in the pericentral zone (Fig. 5B). In addition, collagen staining revealed fibrotic areas in five of eight adult Tbx3 KO livers, while none of the five control animals exhibited fibrosis (Fig. 5C).

Discussion
In contrast to the extensive insight on the role of growth factors that initiate S phase of the cell cycle, little is known about extracellular signals that regulate M phase. The Wnt/β-catenin signaling pathway is one of the mechanisms known to control G1/S transition (35)(36)(37)(38). A role for Wnt signaling during M phase has also been suggested based on high Wnt/β-catenin signaling activity during cell division, through an unknown mechanism (39,40). In this work we have shown that in the liver, M phase is regulated by Wnt signals through the Wnt target transcription factor Tbx3, which in turn represses the mitotic inhibitors E2f7 and E2f8. Interestingly, activating mutations in Wnt signaling components are common in liver cancer (41,42), while E2f7 and E2f8, as well as polyploidy, are known to suppress tumorigenesis (43)(44)(45)(46). These mitotic inhibitors are expressed in several other tissues, such as the placenta and pancreas, where they are also implicated in arresting the cell cycle (8,(47)(48)(49). Whether Wnt signaling likewise regulates mitosis by suppressing E2f7 and E2f8 in other tissues and contexts is relevant to identifying the widespread functions of Wnts in development and disease (41). Our data indicated that Tbx3, acting in the Wnt signaling network, fine tunes the degree and onset of zonated polyploidy in the liver, by promoting mitosis and cell division. Tbx3, which has been shown to act in a dosage-sensitive manner in other contexts (50), seems to be expressed in a gradient in the pericentral zone (SI Appendix, Fig. S1A). Whether there are differences in Tbx3 function or rate of proliferation in TBX3-high cells adjacent to the central vein and TBX3-low cells further into the lobule is unclear. Tbx3-expressing cells may proliferate at different rates or capacities in the context of injury when additional cell cycle inducers promote tissue repair (19). Moreover, Tbx3negative hepatocytes in the periportal zone seem to maintain low ploidy levels through the course of the animal's lifetime (51). Whether mitosis is regulated by a periportal signal in these cells remains to be studied.
While we did not observe major changes in liver weight or morphology due to loss of Tbx3 or increased polyploidy, the fibrotic phenotype of Tbx3 KO mice indicates an important role for cell cycle regulation in the tissue. In the absence of a capacity for cell division, fibrosis becomes a mode of tissue repair and leads to tissue scarring (52,53). Appearance of fibrotic areas in Tbx3 KO livers suggests that loss of Tbx3 is not compatible with long-term homeostatic tissue renewal, likely due to failure in cell division, which leads to compensatory fibrotic repair.
Regarding an absence of growth abnormalities during postnatal development, we hypothesize that the increased polyploidy itself contributes to growth of the tissue and compensates for the lack of cell division, as it has been observed with partial hepatectomy (54). In addition, in the growing and adult liver, Tbx3 is expressed primarily in the pericentral zone, and it is likely that cells from other zones are able to compensate and fill the growth gap in the absence of Tbx3.
Our explorations of the mammalian liver highlight it as a useful and unique model for cell cycle studies. Hepatocytes have distinct modes of cell cycle activity. They first undergo complete cycles and proliferate, then face roadblocks and become polyploid. The temporal regulation of these events provides distinct windows to query the extracellular cues that drive the cell cycle and to understand how these signals are linked to the intrinsic cell cycle machinery.  (58). Alb-CreERT2 mice were a gift from Julien Sage (Stanford University, Stanford, CA) (59). E2f7 f/+ ; E2f8 f/+ were gifted by Alain de Bruin (Utrecht University, Utrecht, The Netherlands) (60) and were rederived at the Stanford Transgenic Facility. Cdh5-CreERT2 mice were used as previously described (61). For knocking out Tbx3 alone or Tbx3; E2f7; E2f8 together using the Axin2-rtTA driver, animals were given 1 mg/mL doxycycline (Sigma D9891) in drinking water from P14 until P28. In experiments involving the Alb-CreERT2 driver, neonatal P3 mice received a single intragastric injection of 0.08 mg tamoxifen (Sigma T5648), dissolved in corn oil with 10% ethanol. For knocking out Wls, Cdh5-CreERT2; Wls f/f mice 8 to 10 wk of age received intraperitoneal injections of tamoxifen on 4 consecutive days and tissues were harvested at 7 d after the last dose of tamoxifen. All mice were housed with a 12-h light/dark cycle with ad libitum access to water and normal chow.

Materials and Methods
CRISPR-Cas9-Mediated Apc Deletion. The adeno-associated virus with a single guide RNA targetting the Apc gene (AAV-sgApc) was produced from the pAAV-Guide-it-Down construct (Clontech Laboratories Inc., 041315) using assembly primers: (forward) 5'CCGGAGGCTGCATGAGAGCACTTG3' and (reverse) 5'AAAC-CAAGTGCTCTCATGCAGCCT3'3. AAV-sgApc contains a U6 promoter and an sgRNA targeting the sequence 5'AGGCTGCATGAGAGCACTTG3' in exon 13 of Apc. Adult CRISPR-Cas9 knockin mice were obtained from JAX and a single intraperitoneal injection of AAV-sgApc was administered at a dose of 1 × 10^13 genome copies/kg. Livers were collected for analysis 4 wk after induction.
Tissue Collection and Processing. Livers were collected, fixed overnight at room temperature in 10% neutral buffered formalin, dehydrated, cleared in His-toClear (Natural Diagnostics), and embedded in paraffin. Sections were cut at 5-μm thickness, deparaffinized, rehydrated, and processed for further staining via immunofluorescence or in situ hybridization assays as described below.
mRNA In Situ Hybridization. In situ hybridizations were performed using the manual RNAscope 2.5 HD Assay-Red Kits (Advanced Cell Diagnostics) according to the manufacturer's instructions. Images were taken at 20× magnification on a Zeiss Imager Z.2 and processed using ImageJ software. Probes used in this study were E2f7 (target region: 612 to 1,526) and E2f8 (target region: 911 to 1,893).
RNA Isolation and qRT-PCR. Liver samples were homogenized in TRIzol (Invitrogen) with Pestle Motor Mixer (Argos Technologies A0001) or bead homogenizer (Sigma). Total RNA was purified using the RNeasy Mini Isolation Kit (Qiagen) and reverse transcribed (High-Capacity cDNA Reverse Transcription Kit; Life Technologies) according to the manufacturer's protocol. qRT-PCRs were performed with TaqMan Gene Expression Assays (Applied Biosystems) on a StepOne-Plus Real-Time PCR System (Applied Biosystems). Relative target gene expression levels were calculated using the delta-delta CT method as previously described (62). Gene expression assays used were Gapdh (Mm99999915_g1) as control, Tbx3 (Mm01195719_m1), E2f7 (Mm00618098_m1), and E2f8 (Mm01204165_g1).
Hepatocyte Isolation and Culture. Hepatocytes were isolated by a two-step collagenase perfusion technique as previously described (31). Six-well plates (Greiner Bio-One 657160) were precoated with collagen (Corning 354236). Primary hepatocytes were plated into 2 mL of expansion media and media were replaced every 2 to 3 d.  Primary hepatocytes were cultured with expansion media till 60% confluent. The Sleeping Beauty System was applied as described in the plasmid DNA transfection protocol from TransIT-X2 Dynamic Delivery System (Mirus Biotech) with modifications. A total of 2.25 μg of pT2-SVNeo-EF1α-eGFP or pT2-SVNeo-EF1α-eGFP-P2A-Tbx3-HA along with the transposase in a ratio of 10:1 were mixed with TransIT-X2 and incubated for 48 h. Then cells were put on G418 selection with expansion media for 48 h. Cells expressing GFP only or GFP together with Tbx3 were expanded for growth curve or flow cytometry analysis.
Hepatocyte Nuclei Isolation and Ploidy Analysis. Hepatocyte nuclei preparation method was developed by modifying the chromatin preparation protocols described previously (63,64). Liver lobes were homogenized in cold 1% formaldehyde in PBS with a loose pestle and dounce homogenizer with 15 to 20 strokes and fixed for 10 min at room temperature. Samples were incubated for 5 min with glycine at a final concentration 0.125 M and centrifuged at 300 × g for 10 min, at 4°C. Pellets were washed in PBS and resuspended with 10 mL cell lysis buffer (10 mM TrisÁHCl, 10 mM NaCl, 0.5% IGEPAL) and filtered through 100-μm cell strainers. A second round of homogenization was performed by 15 to 20 strokes with a tight pestle. Nuclei were pelleted at 2,000 × g for 10 min at 4°C and resuspended in 0.5 mL PBS and 4.5 mL of prechilled 70% ethanol and stored at À20°C for ploidy analysis. Nuclei were resuspended in 5 mL of PBS and approximately 1 million nuclei were stained with 0.5 mL of side-scatter width (SSC-W) / side-scatter height (SSC-H) analysis. Single-stained channels were used for compensation and fluorophore minus one control was used for gating.
Luciferase Assay for Promoter Function. The HSV-TK promoter was cloned upstream of luciferase in the pGL4.10[luc2] plasmid (Promega). A 122-bp region of the E2f7 promoter or a 2.1-kb region of the E2f8 enhancer was cloned into the XhoI site upstream of the HSV-TK promoter. Two T-box binding motifs from the E2f7 promoter were mutated and six motifs were deleted from the E2f8 enhancer. HepG2 cells were cotransfected with 250 ng of the test plasmid and 250 ng of control pRL-TK plasmid, using the TransIT-X system (Mirus). A dual luciferase reporter assay (Promega) was performed at 48 h posttransfection with luminescence detected by a luminometer (Berthold Technologies). Firefly luciferase activity was normalized to Renilla luciferase in each sample.